Viral growth inhibition

ABSTRACT

A synthetic molecule comprises at least one oligonucleotide comprising an RNA binding sequence or sequences corresponding to the site bound by the HIV protein rev and capable of binding ot rev within cells. The binding sequence or sequences, by binding with rev within cells, can act to cause inhibition of growth of any HIV present in the cells, and so has potential therapeutic use in treatment of patients infected with HIV. The invention also provides an assay for identifying that inhibit rev binding.

This is a divisional application Ser. No. 08/540,448, filed Oct. 10,1995, now U.S. Pat. No. 5,786,145, which is a continuation of U.S. Ser.No. 08/030,102, filed Mar. 18, 1993, now abandoned, which is a nationalphase filing of PCT/GB91/01616, filed Sep. 20, 1991, which is a PCTfiling of GB9020541.0, filed Sep. 20, 1990.

FIELD OF THE INVENTION

This invention relates to a method of and compositions for use in theinhibition of viral growth within cells. Specifically, the inventionrelates to the inhibition of growth of the human immunodeficiency virus(HIV).

BACKGROUND TO THE INVENTION

The HIV genome is tightly compressed (FIG. 1). At least 30 different RNAtranscripts are produced by splicing using the six splice acceptors andtwo splice donor sequences [see references 85, 86]. The structuralproteins encoded by HIV are chemically similar to those of the C-typeretroviruses and like them are encoded as polyproteins by the gap (groupantigen), pol (polymerase) and env (envelope) genes. Clevage of thepolyproteins by the viral protease or cellular enzymes generates eightfunctional virion proteins. In addition to these structural genes, HIV-1also caries genes for three regulatory proteins, rev (regulator factor);and two proteins involved in virus maturation, vif (virion infectivityfactor) and vpu (viral protein U). The vpr (viral protein R) geneencodes a low copy number virion component. In the closely relatedviruses HIV-2 and simian immunodeficiency virus (SIV) vpr is replaced byvpx (viral protein X), a unique virion protein.

Transcription of the HIV genome during virus replication shows distinctkinetic phases (see references 53,59,60,79). The initial products of HIVgene expression are short, multiply spliced mRNAs approximately 1.8 to2.0 kb in length, which encode the trans-acting regulatory proteinstat,rev (and possibly nef). As infection by the virus develops, and thelevels of the tat and rev proteins rise in the infected cells, mRNAproduction shifts progressively towards production of a family ofsingly-spliced 4.3 kb mRNAs encoding env and other HIV gene productssuch as vif and vpr. Finally, late in the infection process, productionswitches to full-length, unspliced, transcripts which act both as thevirion RNA and the mRNA for the gag-pol polyprotein.

To achieve this control of gene expression, the HIV virus relies on theinteraction of cellular and virus-encoded trans-acting factors withcis-acting viral regulatory sequences (1,3,53). Initiation oftranscription relies largely on the presence of binding sites forcellular transcription factors in the viral long terminal repeat (LTR)(28). In contrast, the virally encoded regulatory proteins tat and revexert their activity via cis-acting sequences encoded within HIVmessenger RNAs. The trans-activation-responsive region (TAR) is requiredfor tat activity, and is located in the viral long terminal repeat (LTR)between residues +1 and +79 (5,9,10,11,12,13,14,16,27,38). In rev minuscells only the short spliced transcripts appear in the cytoplasm. Ittherefore seemed likely that a regulatory sequence was present in one ofregions removed form regulatory gene mRNAs by splicing. After asystematic search, a cis-acting sequence required for rev activity, wasmapped to a complicated RNA stem-loop structure located within the envrading frame. This sequence has been named the rev-responsive element(RRE). The rev-responsive element (RRE) has been localized to a234-nucleotide long sequence within the env gene (47,51,54,65,67,68,77).Similar regulatory proteins and target sequences are used by HIV-2 andSIV (8,66). The HTLV-1 virus rex gene product appears to functionanalogously to rev, and can functionally substitute for rev to promoteviral gene expression (76).

The distinct kinetic phases of HIV transcription are now believed toreflect the intracellular levels of the regulatory proteins tat and rev.Initially, binding of host transcription factors to the LTR inducedbasal level transcription of the early mRNAs including tat. As tatlevels rise, increased transcription from the LTR is stimulated by thetrans-activation mechanism. This leads to further increases in tatlevels, and also stimulates production of rev. Production of the viralsturctural proteins begins once rev levels have risen to sufficientlyhigh levels to promode export of messenger RNAs carrying therev-responsive element (RRE) sequence. The HIV growth cycle may alsoinclude a latent stage where viral gene expression is silent becausetranscription from the viral LTR produces insufficient amounts ofregulatory proteins to initiate the lytic growth cycle.

Becuase the rev protein acts post-transcriptionally to mediate the shifttowards expression of the late, largely unspliced viral mRNAs(53,60,78,79), rev protein was initially proposed to be a resulator ofsplicing in HIV. Subsequent work has shown that expression of revprotein permits the appearance in the cytoplasm of transcripts carryingRRE sequences. In the absence of rev, mRNAs carrying the RRE sequenceare retained in the nucleus (52,54,65). Although mRNA presursorscarrying heterologous splice donor or acceptor sequences may becomerev-responsive by addition of the intact RRE sequences (65), it is stillunclear whether the effects of the rev protein are coupled to splicingitself or if a still undefined pathway regulates the export of mRNA fromthe nucleus. In vitro, the rates of splicing of strong splice donor andacceptor sequences, such as from the globin gene, appear to beinsensitive to the presence of RRE sequences, suggesting that revfunction competes with the splicing function (46).

Rev recognition of the RRE, like tat recognition of TAR, is due todirect binding [33,34,47,68,73,84]. Binding is tight (K_(d) =1-3 nM) andhighly specific for the RRE [33, 34,84]. However, the binding behaviourof rev to RRE RNA is much more complex than the binding of tat to TARRNA. As the concentration of rev increases, progressively largercomplexes with RRE RNA are formed, whereas tat only forms one-to-onecomplexes with TAR RNA.

The simplest explanation for the RNA binding behaviour of rev is thatthe protein binds initially to a high affinity site and thatsubsequently additional rev molecules occupy lower affinity sites [33].We have recently mapped the high affinity rev binding site to apurine-rich "bubble" located near the 5' end of the RRE [87]. Mutationsthat disrupt or delete the "bubble" abolish RRE activity [51,68]. Thelow affinity binding reaction is the result of both protein-protein andprotein-RNA interactions. At high concentrations rev polymerizes andforms long filaments 14 nm wide and up to 1,500 nm long. Because of itsability to polymerize, when rev is mixed with HIV mRNAs the RNA ispackaged into rod-like ribonucleoprotein filaments. Filament assemblyappears to be nucleated by the binding of rev to the RRE and is muchmore efficient on RNA molecules carrying a functional RRE sequence thanon molecules that do not include an RRE sequence than on molecules thatdo not inlcude an RRE sequence [87].

The RNA binding properties of rev have led us to propose that rev blockssplicing simply by packaging unspliced RNA transcripts containing theRRE sequence into inaccessible ribonucleoprotein complexes [87].Although complexes containing rev and viral mRNAs have not yet beenisolated from infected cells, there is already indirect evidence insupport of this type of mechanism. For example, it is believed that theblocking of splicing in vitro by rev is due to the disruption ofspliceosome assembly [46]. Furthermore, the in vivo activity of revappears to be highly concentration dependent, as would be expected for amechanism of action based on RNA packaging. Rev-minus viruses can onlybe rescued by co-transfection with very high levels of rev-expressingplasmids [88].

The packaging model also provides a simple kinetic explanation for thedelayed appearance of the virion RNA and physical explanation for howrev can act on RREs placed in a wide variety of positions. During HIVinfection high levels of the 4.3 kb mRNAs, such as the env mRNA, aresynthesized for several hours before significant levels of thefull-length virion RNA is produced [59]. Compared to the 4.3 kb mRNAs,the virion RNA carries additioknal unused splice donor and acceptortowards its 5' end, far away from the RRE where filament formation issuggested to nucleate. If stabilization of the virion RNA requiresproduction of a longer ribonucleoprotein filament than the stabilizationof the 4.3 kb mRNAs, it is easy to imagine that this would only takeplace late in the infectious cycle, when intracellular rev proteinconcentrations are expected to be maximal.

Although we believe tha the physical properties of rev can alone accountfor its biological activity, there have been some reports that cellularco-factors(s) are also required [89]. Trono and Baltimore suggested thata human cell contains a species-specific factor required for revactivity after observing that mouse cells infected by HIV have arev-minus phenotype which can be easily reversed by fusion to humancells [89]. However, it is possible that rev protein levels differedbetween the various cell lines, and that only sub-threshold levels ofrev were expressed in the mouse cells [89]. By contrast, rev isfunctional in Drosophila melanogaster cells [90].

It is an aim of the present invention to provide an effective methodfor, and compositions for use in, the inhibition of HIV viral growthwithin cells, which involves modifying the activity of the regulatoryprotein rev in the viral growth cycle, and also an assay for screeningpotential anti-viral agents.

STATEMENT OF THE INVENTION

According to a first aspect of the present invention there is provided asynthetic molecule comprising at least one oligonucleotide comprising anRNA binding sequence or sequences corresponding to the site bound by theHIV protein rev and capable of binding to rev within cells.

The binding sequence or sequences, by binding rev within cells, can actto cause inhibition of the growth of any HIV present in the cells, andso has potential therapeutic use in treatment of patients infected withHIV.

According to a second aspect of the present invention, there is provideda pharmaceutical composition comprising a molecule as provided by thefirst aspect of the invention. The pharmaceutical composition isconveniently for use as an inhibitor of growth of HIV within cells.

According to a third aspect of the invention, there is provided asynthetic molecule for use in treating patients infected with HIV, themolecule comprising at least one oligonucleotide comprising an RNAbinding sequence or sequences corresponding to the binding site bound bythe HIV protein rev and capable of binding to rev within cells.

According to a fourth aspect of the invention, there is provided the useof a molecule as provided by the first aspect of the invention, in themanufacture of a medicament for inhibiting growth of HIV within cells.

The present invention is based around the unexpected discovery that onlya small and specific region of the RRE sequence is critical for bindingrev protein. It is therefore reasonably practicable to synthesise(chemically or enzymatically) pharmaceutically acceptable nucleic acidor other analogues of this specific binding site, which are sufficientlysmall to be capable of assimilation into cells.

These analogues can then be used as inhibitors of rev protein activityin cells; by binding to rev present in the cells the analogues are ableto block its reaction with the RRE present on viral transcripts and thusviral growth in those cells could be effectively inhibited.

It has been found that transcripts corresponding to RRE residues 26-96,33-96, and 26-66 bind rev with the same affinity as the full-length 223nucleotide transcript. However, transcript 37-96 has no discernibleaffinity for rev. Therefore the rev binding sequence maps to betweennucleotides 33 at the 5' end and 66 at the 3' end. Thus, the maximumsize of the rev binding site is 34 nucleotides and this corresponds to apredicted stem-loop structure which is necessary (and may be sufficient)for rev binding and RRE function in vivo. It has further been found thatrev recognizes a specific purine rich "bubble" formed by these stem-loopstructures. Placement of the "bubble" in other non-homologous stem loopstructures confers specificity for rev binding. Chemically synthesizedanalogues of the rev binding site, containing the purine-rich "bubble"structure are also able to bind rev spefically and to competeeffectively with the full-length RRE sequence.

Hence, the binding sequence in the oligonucleotide of the presentinvention preferably comprises a sequence ofnon-Watson-Crick-base-paired residues corresponding to those in theHIV-1 RRE fragments which form this "bubble" structure.

Because such a small region of the RRE is actually needed to bind rev,analogues of the binding site can be constructed which are composed ofoligonucleotides of therapeutically useful lengths, preferably (but notlimited to) twenty residues or less. Such molecules are more likely tobe able to enter infected cells, and hence to be of use inpharmaceuticals for in vivo treatment of HIV infections, than are thoseof greater length. A molecule in accordance with the invention is thuspreferably in the form of an oligonucleotide(s) less than or equal totwenty residues in length, so as to facilitate assimilation into cellsinfected with HIV.

Since RNA itself is metabolically unstable in cells, theoligonucleotide(s) used in of the present invention is preferablymodified in some way so as to increase its stability in the cells. Thebinding sequence, which is necessarily an RNA sequence, may for instancebe incorporated into a DNA basic sequence or some otherstructurally-related variant oligonucleotide, which basic sequenceimparts the necessary metabolic stability to the oligonucleotide(s) as awhole.

Thus any oligonucleotide (or combination of oligonucleotides) whichincludes the RNA sequence corresponding to the site bound by rev, whenintroduced into cells, should be capable of binding rev and thus actingas a competitive inhibitor of viral growth within those cells. Indeed,any small molecule which is able to bind to rev at the RRE RNA bindingsite could be used as an anti-viral agent, and the invention includeswithin its scope such a molecule for use as an anti-HIV agent. Such amolecule could mimic the shape of the RNA structure in RRE RNA or couldcontain functional groups equivalent to the RNA structure in RRE RNA.

Since oligoribonucleotides are sensitive to cleavage by cellularribonucleases, it may be preferable to use as the competitive inhibitora chemically modified oligonucleotide (or combination ofoligonucleotides) that mimics the action of the RNA binding sequence butis less sensitive to nuclease cleavage. Other modifications may also berequired, for example to enhance binding, to enhance cellular uptake, toimprove pharmacology or pharmacokinetics or to improve otherpharmaceutically desirable characteristics.

The oligonucleotide may be a naturally occuring oligonucleotide, or maybe a structurally related variant of such an oligonucleotide havingmodified bases and/or sugars and/or linkages. The term "oligonucleotide"as used herein is intended to cover all such variants.

Modifications, which may be made either to the binding site per se or tothe part of the oligonucleotide not involved in binding, may include(but are not limited to) the following types:

a) Backbone modifications (see FIG. 4 below)

i) phosphorothioates (X and Y or W or Z═S or any combination of two ormore with the remainder as 0).

e.g. Y═S (81), X═S (49), Y and Z═S (45)

ii) methylphosphonates (eg Z=methyl (69))

iii) phosphoramidates (Z=N-(alkyl)₂ e.g. alkyl=methyl, ethyl, butyl)(Z=morpholine or piperazine) (44) (X or W═NH) (64)

iv) phosphotriesters (Z═O-alkyl e.g. methyl, ethyl etc) (70)

v) phosphorus-free linkages (e.g. carbamate, acetamidate, acetate)(55,56)

b) Sugar modifications

i) 2'-deoxynucleosides (R═H)

ii) 2'-O-methylated nucleosides (R═OMe) (80)

iii) 2'-fluoro-2'-deoxynucleosides (R═F) (61)

c) Base modifications--(for a review see 58)

i) pyrimidine derivatives substituted in the 5-position (e.g. methyl,bromo, fluoro etc) or replacing a carbonyl group by an amino group (75).

ii) purine derivatives lacking specific mitrogen atoms (eg 7-deazaadenine, hypoxanthine) or functionalised in the 8-position (e.g. 8-azidoadenine, 8-bromo adenine)

d) Oligonucleotides covalently linked to reactive functional groups,e.g.:

i) psoralens (71), phenanthrolines (82), mustards (83) (irreversiblecross-linking agents with or without the need for co-reagents)

ii) acridine (intercalating agents) (57)

iii) thiol derivatives (reversible disulphide formation with proteins)(48)

iv) aldehydes (Schiff's base formation)

v) azido, bromo groups (UV cross-linking)

vi) ellipticenes (photolytic cross-linking) (74)

e) Oligonucleotides covalently linked to lipophilic groups or otherreagents capable of improving uptake by cells, e.g.:

i) cholesterol (63), polyamines (62), other soluble polymers (e.g.polyethylene glycol)

f) Oligonucleotides containing alpha-nucleosides (72)

g) Combinations of modifications a)-f)

It should be noted that such modified oligonucleotides, while sharingfeatures with oligonucleotides designed as "anti-sense" inhibitors, aredistinct in that the compounds correspond to sense-strand sequences andthe mechanism of action depends on protein-nucleic acid interactions anddoes not depend upon interactions with nucleic acid sequences.

The molecule and the pharmaceutical composition of the present inventionmay be administered orally, intravenously or by any other suitablemethod when used to inhibit viral growth in vivo. They may also be usedto inhibit viral growth in vitro, for instance in cells in blood whichhas been removed from a living organism and is later required fortransfusion purposes.

The present invention further provides a method of inhibiting growth ofHIV virus within cells, comprising the step of administering to thecells a molecule or pharmaceutical composition in accordance with theinvention.

The invention also provides a method of treatment of a patient infectedwith HIV, comprising the step of administering to the patient a moleculeor pharmaceutical composition in accordance with the invention.

The invention can also be used as the basis of an assay for identifyingcompounds that inhibit binding of rev protein to RRE RNA or syntheticanalogs thereof, and so have potential use as anti-viral agents.

Thus in a further aspect the invention provides an assay for identifyingcompounds that inhibit binding of rev protein to RRE RNA, comprisingreacting a compound with rev protein and a molecule in accordance withthe invention, and determining the degree of binding of rev to themolecule.

By determining the degree of binding of rev to the molecule, andcomparing this with results for known standards, an indication can beobtained of the degree of inhibition (competitive or non-competitive) ofrev/RRE binding caused by the compound.

The assay is preferably in the form of a filter binding assay, but mayalso be another type of assay such as a gel mobility-shift assay, aspectroscopic assay, capture assay etc.

Accurate measurement of the affinity of rev for RRE RNA in the presenceof inhibitor molecules, such as competitor molecules that compete forbinding to RRE or molecules that inhibit rev binding to RREnon-competitively, cannot be achieved without the formation ofstoichiometric complexes between tat protein and RRE RNA, which is nowpossible in vitro as a result of improvements in the purification of revprotein from E. coli and in methods for performing binding assays.

Compounds identified as having an inhibitory effect on rev/RRE bindingcan then be further investigated for possible use as an anti-viralagent. The invention can thus enable screening of potential anti-viralcompounds.

The present invention will now be described in greater detail, by way ofillustration, with reference to the accompanying Figures, of which:

FIG. 1 illustrates the genetic elements and cellular factors controllingHIV-1 gene expressiuon;

FIG. 2 shows how the RNA-binding protein rev may control HIV-1 geneexpression;

FIG. 3 shows the structure of the rev-response element RNA foldedaccording to the program of Zuker (SEQ ID NO: 1);

FIG. 4a shows the predicted secondary structure of the rev-responseelement RNA as given incorrently by Malim et al. (65) while

FIG. 4b shows the correct structure predicted using the program ofZuker. The Figure also indicates sites of enzymatic cleavage andchemical modification as determined by Kjems et al. (92);

FIG. 5 illustrates rev binding to RRE fragments and shows the predictedsecondary structures for RNA transcripts spanning selected regions ofthe rev-response element;

FIG. 6 is a graph of fraction bound versus rev concentration (nanomolar)and shows saturation binding curves for rev binding to RNA transcriptsspanning selectd regions of the rev-response element;

FIG. 7 shows the structures of modified oligonucleotides which mightshow potential anti-viral activity;

FIG. 8a shows a Scatchard analysis of rev binding to a 238 nucleotidelong transcript of the rev response element.

FIG. 8b shows the same data plotted as a double-reciprocal plot;

FIGS. 9a-j show the structures of short RNA transcripts carrying a highaffinity rev binding site consisting of the purine-rich "bubble";

FIG. 10 is a graph of fraction RNA bound versus rev proteinconcentration (nM) and shows saturation binding curves for rev bindingto RNA transcripts carrying a series of mutations in the purine-rich"bubble". The mutations are listed in Table 1;

FIG. 11 is a graph of fraction RNA bound versus RNA competitorconcentration (nM) and shows competition binding curves for rev bindingto RNA transcripts carrying a series of mutations in the purine-rich"bubble";

FIG. 12 shows the structures of chemically synthesizedoligoribonucleotides which when annealed form the binding site for rev(boxed);

FIG. 13 is a graph of fraction of 32P labelled RRE RNA R7 bound versuscompetitor RNA concentration (nM), and shows a competition filterbinding assay using the chemically synthesized oligonucleotides shown inFIG. 12 (RBC4 (filled diamonds), RBC5 (filled squares with dots), RBC6(filled diamonds with dots)) as well as an enzymatically synthesizedtranscript corresponding to a 238-nucletotide long RRE sequence (R7)(empty squares with dots);

FIG. 14 shows a Scatchard plot of the binding of the chemicallysynthesized oligonucleotide RBC6 to rev. The structure formed by RBC6binds approximately 4 molecules to rev protein with linear concentrationdependence and a binding constant of approximately 0.3 nM;

FIG. 15 to a graph of retention of radioactivity versus rev (nM), andshows the saturation binding curve of rev protein binding to thechemically synthesized oligonucleotide RBC6;

FIG. 16 shows the structures of chemically synthesizedoligoribonucleotides which when annealed form a three-way junction thatstabilizes the binding site for rev (boxed);

FIG. 17 is a graph of fraction of hot (³² P labelled RRE RNA) R7 boundversus competitor (nM) and shows a competition filter binding assayusing the annealed, chemically synthesized oligonucleotides shown inFIG. 16 (TWJ-C, TWJ-U, RBC2-6X); and

FIG. 18a shows an electron micrograph of the filament structures formedby rev protein alone,

FIG. 18b shows an electron micrograph of the filament structures formedby rev protein in the presence of 238-nucleotide long RRE RNAtranscripts, and

FIG. 18c shows an electron micrograph of the filament structures formedby rev protein in the presence of 2400-nucleotide long transcripts ofthe env mRNA, carrying an RRE sequence towards its 3' end.

DETAILED DESCRIPTION OF THE DRAWINGS

In FIG. 1 the mechanisms for control of HIV gene expression by theregulatory proteins tat and rev is shown schematically. In newlyinfected cells, binding of cellular transcription factors to the longterminal repeat (LTR) stimulates a basal level of transcription of theearly mRNAs encoding tat, rev and nef (shown in panel 1). As tat levelsrise in the cell, transcription is stimulated by the trans-activationmechanism. This leads to increased production of the early mRNAs andaccumulation of partially spliced or unspliced RNA transcripts in thenucleus (panel 2). As rev levels rise in the cell, RNAs carrying therev-response element are stabilized and exported from the nucleus. Theselate mRNAs act as messengers for the structural proteins encoded by thegag, pol and env genes. The full-length HIV transcript acts both as amRNA for gap-pol and as the virion RNA (panel 3).

FIG. 2 shows a more detailed model for control of HIV gene expression byrev. In the absence of rev, spliceosome formation and splicing isefficient (panel a). As rev levels rise in an infected cell, the proteinbinds to RNAs carrying the rev-responsive element, but there isinsufficient protein available to effect spliceosome formation (panelb). We imagine that after a monomer or small aggregate of rev binds tothe high affinity site in the RRE, additional molecules of rev couldco-assemble along the length of the mRNA precursors by virtue ofprotein-protein and lower affinity protein-RNA interactions. Thus, athigh rev concentrations RNA transcripts carying the RRE are packagedinto filamentous structures (panel c). As a result of filamentformation, splicing is blocked and unspliced mRNAs are exported from thenucleus. As discussed below, filament formation by this mechanism couldaccount for the effects of rev on the processing of viral mRNAs. Thismechanism is consistent with the chimical properties of rev as describedbelow as well as the observations that the RRE is functional when placedeither in an intron or in an unspliced exon (65) and is consistent withthe suggestion of Change & Sharp (46) that rev disrupts spliceosomeassembly. Our model also suggests that mutants of rev that blockfilament formation in vitro will be dominant in vivo. A number ofdominant mutations of rev have already been described (67,91), and itwill be of interest to determine whether these affect filamentformation.

FIG. 3a shows the secondary structure for the RRE region ofHIV-1_(ARV-2) (residues 7786 to 8010) predicted by the RNA foldingprogrammes of Zuker (14) SEQ ID NO: 1. Residue 1 of the RRE is the firstnucleotide of the StyI site used originally to define the location ofthe RRE sequence (65). This corresponds to residue 12 according thenumbering system of Kjems et al. (92). Our model differs from that ofMalim et al. (65), because the pairing of U33, G34 and G65 with A63, C62and U85 respectively allows formation of the purine-rich "bubble". Basepairing between residues A113 to U118 and A181 to U186 is allowed forthe HIV-1_(ARV-2) sequence shown above, but this feature is absent inthe HIV-1_(HXB2) sequence analysed by Malim et al. (65).

In panel b, the structure of the purine-rich bubble seuqnce is shownwith individual bases numbered.

FIG. 4 compares the predicted secondary structure of the rev-responseelement RNA (SEQ ID NO: 2) as originally proposed by Malim et al. (65)with the corrected structure predicted using the program of Zuker. TheFigure also indicates sites of enzymatic cleavage and chemicalmodification as determined by Kjems et al. (92): empty circles indicatekethoxal, filled circles indicate DEP, arrows with filled heads indicateRNase T2 and arrows with empty heads indicate RNase CV1. A complicatedstem-loop structure for RRE RNA has been proposed by Malim et al. basedon the RNA folding programs of Maizel (65,93). However, the RNA foldingprograms of Zuker (94) predict that a more stable structure could beformed by including the pairing of U33 and G34 with C62 and A63 on onestem and G65 with U85 respectively on an adjacent stem. As shown in theFigure, these base pairs create a purine-rich "bubble".

The new structure is more consistent with the nuclease-protection andchemical probing data reported by Kjems, et al. (92) than the modelproposed by Malim et al. (65). For example, G59, G64 and G65 arestrongly modified by kethoxal whereas the residues G34, G35, G36 areonly weakly modified and none of these residues is susceptible tocleavage by ribonuclease T1. In the new structure, G34, G35, G36 arestacked on one side of the bulge, whereas G59, G64 and G65 appear to bemore accessible. In the original model, all these residues appeared in alarge open loop. Furthermore, A32 and A86, which are readily modified bydiethylpyrocarbonate (92), now appear as bulged residues whereas in theoriginal proposal these residues were base-paired.

FIG. 5 shows the predicted secondary structures for RNA transcriptsspanning selected regions of the rev-response element. These include RREfragments 1-96, 26-92 and 26-72. Fragments corresponding to each of thetranscripts were cloned into a pGEM vector and transcribed from HindIIIlinerise DNA using T7 RNA polymerase. The sequence GGGAGACCGGAAUUC [SEQID NO: 2] on the 5' end of each sequence was contributed by the vector.At the 3' end of each transcript a single A residue was contributed bythe vector.

From FIG. 5 it can be seen that RRE fragments 1-96 (SEQ ID NO: 3) and26-92 (SEQ ID NO: 4) form similar "bubble" structures in the boxedregions beginning with residue U-26. Both of these fragments bind revwith the same affinity as the full-length RRE sequence. Fragment 26-72(SEQ ID NO: 5) includes the same sequences but does not form the samesecondary structure, and in consequence, does not bind rev. Hence, notonly the base sequence but also the structure represented by the"bubble" exhibited in fragments 1-96 and 26-92 is critical for revbinding activity.

FIG. 6 shows satruation binding curves for rev binding to a series ofRNA transcripts spanning selected regions of the rev-response element.The "sense" sequence (indicated by empty squares with dots) is thefull-length RRE sequence. Identical binding behaviour was observed usingfragments including residues 26-96 (filled squares with dots), 26-92(filled diamonds with dots), 26-66 (filled squares) and 33-96 (emptysquares). However, "antisense" sequences (filled diamonds) carrying thecomplementary sequence to the RRE and a transcript from 37 to 96 (filledtriangles) failed to bind rev efficiently. Therefore the rev bindingsequence must map to between nucleotides 33 at the 5' end and 66 at the3' end. Thus, the maximum size of the rev binding site is 34nucleotides, and this corresponds to the predicted stem-loop structurewhich is necessary (and may be sufficient) for rev binding and RREfunction in vivo.

These experiments strongly suggest that nucleic-acid analogues of thebinding site for the regulatory protein rev are potential competitiveinhibitors of regulatory protein activity in vivo. For example, a shortnucleic acid seuqnece corresponding to the region of RRE RNA betweenresidues 33 and 66 is capble of binding rev with an affinity similar tothat of the complete RRE sequences. As described below, chemicallysynthesized sequences of even shorter length are capable of binding revand hence inhibiting viral growth, provided that they are able to formsuitable stem-loop structures.

Method

Rev protein was expressed and purified as described (33) or furtherpurified by chromatography on Heparin-Sepharose. Rev was applied toHeparin columns in a buffer containing 220 mM NaCl/50 mM Tris-HClpH8.0/1 mM DTT/0.1 mM EDTA 0.1% Triton X-100 and eluted in buffercontaining 2 M NaCl. After gel filtration on Superose 6 (prep grade)columns equilibrated with 200 mM NaCl/50 mM Tris-HCl pH8.0/1 mM DTT/0.1mM EDTA, the rev protein appeared homogeneous on SDS-polyacrylamide gelsand free of RNA contaminants. Rev concentrations were determined byamino acid analysis of the purified protein.

DNA inserts containing RRE-related sequences were cloned between theEcoRI (5') and HindIII (3') sites of pGEM 1, either by cloning PCRproducts or by cloning annealed pairs of synthetic oligonucleotides. RNAtranscripts for binding experiments were prepared by transcription ofHindIII cut plasmids using T7 RNA polymerase and purified by gelelectrophoresis. These transcripts carry a 5' extension of 15nucleotides contributed by the vector and an extra A residue at the 3'end from the HindIII site, as described above.

For filter binding assays, each reaction mixture contained 20 pg ofuniformly labelled RNA probe (approximately 500 cpm/pg RNA), 1 mgsonicated salmon sperm DNA, 0.45 mg yeast tRNA, 40 units RNasin(Promega) in 500 ml TK buffer (43 mM Tris-HCl pH8.0, 50 mM KCl).Incubation was at 4° C. for 15 minutes in the presence of 0 to 10 nMpurified rev protein. In some experiments, other reagents, such as 16SrRNA were added to the binding reactions as indicated in the Figurelegends. To measure binding, each reaction mixture was applied undergentle vacuum to a 0.45 um Millipore filter which had been pre-wettedwith TK buffer. The filters were washed with 3×600 ml TK buffer, dried,and radioactivity counted by liquid scintillation.

FIG. 7 shows the structures of an oligonucleotide which could be of usein the synthesis of RNA fragments with antiviral activity. Modificationsto the oligonucleotide, in order to decrease its sensitivity to nucleasecleavage, or otherwise to increase its stability may include alterationsto the structures of the bases B1 and B2, the sugar backbone R or thephosphate linkages W, X, Y and Z, as described above.

FIG. 8 shows a Scatchard analysis of rev binding to a 225nucleotide-long RRE RNA sequence. We have reported previously that asthe concentration of rev increases, progressively larger complexes withRRE RNA are formed, whereas rev is unable to form stable complexes withanti-sense RRE and other RNA sequences (33). This experiment, which hasrecently been repeated by others (92), strongly suggested that rev bindsinitially to a high affinity site on the RRE and that subsequentlyadditional rev molecules occupy adjacent sites. We have now shown thatthese additional rev molecules bind to the RRE RNA with lower affinity.As shown in FIG. 8a, the Scatchard plot for rev binding to RRE RNA isnon-linear, whereas a protein which forms one-to-one complexes with RNA,such as tat, produces a linear Scatchard plot (95). In FIG. 8b the samedata is plotted as a double-reciprocol plot.

We have estimated that the K_(d) for high affinity rev binding by alinear regression analysis of the high affinity data. At 50 mM KCl (vgreater than 28) there is a site to which rev binds with an apparentK_(d) of 2±0.6 nM (50 mM KCl). At 200 mM KCl (v greater than 10) theK_(d) for high affinity binding is 4±1.0 nM. Both these values areconsistent with previous estimates of a K_(d) of between 1.0 and 3.0 nMobtained from saturation binding experiments (33,34). However, it shouldbe noted that estimates of K_(d) by any simple binding experiment thatuses labelled RNA as a probe will include the contributions of both thehigh affinity site and the adjacent lower affinity sites (96).

The stoichiometry of rev binding to RRE RNA is highly dependent on ionicstrength. At 50 mM KCl between six and eight rev monomers bind to theRRE RNA, whereas the stoichiometry of binding is approximately 2:1 at200 mM KCl (FIG. 1b). In agreement with previous reports (11), we havefound that rev elutes from gel filtration columns equilibrated with 200mM NaCl with an apparent mass of 60 kDa (data not shown). These resultssuggest that rev exists in solution as a small oligomer, most likely atetramer (11,12), that is able to bind to RNA.

Method

Binding reactions were performed in buffers containing 50 mM Tris-HClpH8.0, 1 ug sonicated salmon sperm DNA, 0.45 ug of yeast tRNA and 20units of RNAsin (Promega) and either 200 mM KCl (filled circles) or 50mM KCl (filled squares). Reactions at 200 mM KCl contained 9 to 10 nMrev and between 1 to 90 nM RRE RNA. Reactions at 50 mM NaCl contained 30to 34 nM rev and between 1 to 90 nM RRE RNA. In FIG. 8a the data isplotted as the stoichiometry, v (the ratio of the concentration of boundRNA to rev protein; abscissa) versus the ratio of v to the free RNAconcentration (ordinate). In FIG. 8b, the data is plotted as thereciprocal of the stoichiometry versus the reciprocal of the free RNAconcentration.

FIG. 9 shows representative short RNA stem-loop structures assayed forrev-binding. The sequences shown represent the full transcriptsincluding residues derived from vector sequences. The optimal structurespredicted by the method of Zuker (94) are shown, with the purine-rich"bubble" boxed. (a) Transcript R19 (SEQ ID NO: 6) contains RRE sequences33 to 66. (b) R22 (SEQ ID NO: 7), residue A56 is deleted from R20. (c)R29 (SEQ ID NO: 8), residues are altered in the apex of the loop to givethe anti-sense sequence. (d) R30 (SEQ ID NO: 9) base pairing in theupper stem structure is altered to give the anti-sense sequence. (e) R38(SEQ ID NO: 10), the stem-loop structure C39 to A56 is replaced by thestable loop sequence CUUCGG (15). (f) R33 (SEQ ID NO: 11), the stem-loopformed by residues 33 to 63, with residue A56 deleted, was inserted ontop of a stem containing 9 base pairs. (g) R37 (SEQ ID NO: 12), thepurine-rich bubble is replaced by an altreed sequence carrying bulged Aresidues on the 5' side. (h) R35 (SEQ ID NO: 13), the purine-rich bubbleis replaced by an altered sequence carrying a single bulged G residue onthe 5' side. (i) R36 (SEQ ID NO: 14), the bulged residues G59-A61 aredeleted. (j) R34 (SEQ ID NO: 15), the bulged residues G35, G36 aredeleted.

As described above the high affinity rev binding site is located betweenresidues A26 and A96. The shortest T7 transcript that is able to bindrev with a K_(d) of 1-3 nM includes RRE sequences beginning at U33 andending at C66 (R19.) This sequence is predicted to fold into the stablestem-loop containing the purine-rich "bubble" shown in FIG. 3. Thestructure is stabilized by a four-base pair stem below the "bubble"which contains CU residues derived from the T7 leader sequence.

Only the boxed sequences in the "bubble" region are required for revbinding. Deletion of the bulged A, at residue 56 (R22) or replalcementof the entire upper stem sequence with the stable RNA hairpin loopsequence CUUCGG (R38) produced transcripte which bound rev with K_(d)=1-3 nM. Similarly, replacement of the upper stem and loop by antisensesequences (R20, R30) resulted in transcripts with normal rev bindingactivity.

Normal rev binding was also observed when the purine-rich "bubble" wasinserted into an elongated RNA stem-loop structure (R33). The bulgedresidues on both sides of the purine-rich "bubble" are required forspecific rev binding. Deletion of G59, U60 and A61 created a structurewith a G, G bulge on one side of the helix (R36) and resulted on loss ofrev binding. Deletion of G35 and G36 from the other side of the helix(R34) or replacement of these residues with bulged A residues (R37) alsoabolished specific rev binding. Deletion of G35 alone only reduced revbinding slightly (R35).

FIG. 10 shows saturation binding curves for rev binding to RNAtranscripts carrying a series of mutations in the purine-rich "bubble".The truncated RRE RNA transcripts containing the rev binding site canbind to rev with a dissociation constant similar to that of full-lengthRRE (i.e. K_(d) =1-3 nM). Because the R33 RNA is in a very stablepredicted (conformation delta G•=-29.6 kcal/mol), we were able tointroduce deletions and substitutions within the purine-rich "bubble"region of R33 RHA without disrupting its overall structure (Table 1).

The results are represented as follows:

    ______________________________________                                        R7              empty square with dot                                           R33 filled diamond                                                            R34 filled square with dot                                                    R44 filled diamond with dot                                                   R49 filled square                                                             R50 empty square                                                              R52 filled triangle                                                           R53 filled triangle with dot                                                  R54 filled square                                                             R55 cross                                                                     R57 empty square with cross                                                 ______________________________________                                    

Nucleotide substitutions (Table 1) are tolerated at the U60 residue(R52), and a G36 (R50) but deletions or substitutions affecting theother bulged residues resulted in complete loss of specific rev binding.For example, replacement of G35 with A (R47) or A61 with G (R53)abolished rev binding.

The four base pairs immediately adjacent to the bulged residues in thepurine-rich "bubble" are also important for rev binding (Table 1).Replacement of C37:G58 with a G:C base pair (R41) or replacement ofG34:C62 with a C:G base pair (R40) abolished specific rev binding.Alterations to the other base pairs in the "bubble" region also reducedrev binding significantly (Table 1). The only neutral mutation that wediscovered was in R57, in which the base pair G38:C57 is replaced by anA:U base pair.

FIG. 11 shows competition binding curves for rev binding to RNAtranscripts carrying a series of mutations in the purine-rich "bubble".unlabelled RRE RNA was an effective competitor and reduced rev bindingto the labelled RRE RNA by 50% of the initial value with a D_(1/2) =2nM. The short R33 transcript was also an effective competitor andreduced rev binding with D_(1/2) =8 nM. The mutations in the "bubble"either reduce or abolish specific rev binding. For example, R34, whichcarries a deletion of the bulged G residues G35 and G36, does not bindrev with measurable affinity and did not compete efficiently for revbinding against the RRE (D_(1/2) greater than 250 nM). R35 which has abulge containing a single residue, and is typical of a mutation withreduced rev affinity had a K_(d) of 4 nM and showed intermediatecompetition behaviour (D_(1/2) =16 nM).

Method

Filter binding reactions contained 17 nM rev, 0.5 pM labelled RRE RNAand between 0-100 nM unlabelled competitor RNA. Empty squares, RRE RNAcompetitor; filled circles, R33 RNA competitor; filled triangles, R34RNA competitor; empty triangles, R35 RNA competitor.

The binding of chemically synthesized RNA by rev protein (FIGS. 12-15).

FIG. 12 shows the structures of small RNA duplexes containing apurine-rich bubble. Boxed residues are those which have been shown to beessential for specific recognition by rev protein: RBC4 (RBC is anabbreviation for Rev Binding Core) has 4 base pairs in the stem on eachside of the bubble (SEQ ID NO: 16 and SEQ ID NO: 17). Similarly RBC5 has5 base pairs (SEQ ID NO: 18 and SEQ ID NO: 19) and RBC6 has six basepairs on each side (SEQ ID NO: 20 and SEQ ID NO: 21). Theoligoribonucleotides were synthesised chemically essentially asdescribed (1) and purified by reversed-phase HPLC. When annealedpairwise the oligonucleotides can form duplexes containing therecognition site for tat protein.

FIG. 13 shows the results of a competition filter binding assay tocompare the binding to rev of the chemically synthesized duplexes withthat of a 238-base RRE transcripts (R7). In these experiments, uniformly³² P-labelled R7 (1.5 nM) was competed against 0-60 nM unlabelledcompetitor RNA in the presence of 24 nM rev protein. The results showthat RBC6, which has 6-bp stems, competed about half as well as R7,while RBC5 and RBC4 had less and no competitive binding abilitiesrespectively. These results demonstrate that small duplexes consistingof chemically synthesized oligonucleotides can bind to rev protein andact as inhibitors of rev.

FIG. 14 shows a Scatchard analysis of the binding of RBC6 to rev.Binding experiments were carried out using labelled RBC6 duplex formedfrm the 5'--³² P-labelled 14-mer oligoribonucleotide annealed to the 15mer (0.6 nM) and 0-150 nM unlabelled RBC6 in the presence of 24 nM revprotein. The results show that the stoichiometry of protein: RNA isapproximately 4:1 and that the dissociation constant (K_(d)) isapproximately 3 nM, a value very similar to that for full length RREinteraction with rev. Also the linearity of the graph shows that thereare no lower affinity binding sites for rev protein on RBC6 in contrastto full length RRE which shows a non-linear Scatchard plot. FIG. 15shows the result of saturation binding experiments using 1.5 nM ³² Plabelled RBC6 and 0-50 nM rev. From these data, a Scatchard plot gave astraight line with a K_(d) value of 3.8 nM.

Method

a) Chemical Synthesis of Oligoribonucleotides

This was carried out on an Applied Biosystems 380B Synthesizeressentially as described (100) using protected ribonucleoside3'--phosphoramidites purchased from MilliGen. After deprotection, theoligoribonucleotides were purified by reversed-phase HPLC using auBONDAPAK C18 column (7.8 mm×300 mm) with a linear gradient ofacetonitrile in 0.1M triethylammonium acetate buffer (pH7.0). The purityof each oligonucleotide was analysed by anion-exchange HPLC using aHICHROM P10SAX column (10 mmID×250 mmL) with a linear gradient ofpotassium phosphate (pH6.3) in formamide-water (3:2, v/v). Afterdesalting using a Sephadex NAP-10 column (Pharmacia), two appropriateoligonucleotides were annealed by mixing (typically 500 pmol each in 50ul of water) by heating at 90° C. for 2 min and by cooling gradually toroom temperature.

b) Preparation of Labelled and Unlabelled R7 transcript

The mixtures for transcription reaction (500 ul for ³² P-labelled and100 ul for unlabelled) included 0.1 mg/ml template HindIII cut plasmidDNA, 32 mM Tris-HCl (pH7.4), 20 mM NaCl, 13 mM MgCl₂, 8 mM DTT, 2.4 mMeach of ATP, GTP, CTP and UTP, 800 units/ml RNasin (Promega) and 1-15units/ul T7 RNA polymerase. For radiolabelling, 100 uCi of (alpha-³² P)UTP was added. The reaction was allowed to proceed for 2 hr at 37° C.and stopped by addition of EDTA to 30 mM. After phenol extraction, thesolution was concentrated by butanol extraction, and the 238-basetranscript was purified by 6% polyacrylamide gel electrophoresisfollowed by elution with 0.5M ammonium acetate, 1 mM EDTA (pH7.4) and0.5% sodium dodecylsulfate, butanol extraction and ethanolprecipitation.

c) Competition Assay

The mixtures for competition binding (0.5 ml) included 1.5 nM ³²P-labelled R7 (ca. 20,000 cpm), 24 nM rev protein, 1.5 ug/ml calf thymusDNA, 0.67 ug/ml yeast rRNA, varying concentrations (0-60 nM) ofunlabelled competitor RNA and 80 units/ml RNasin in TK buffer (50 mMTris-HCl (pH7.9) and 20 mM KCl). Binding was allowed to take place formore than 15 min on ice, and each mixture was passed through a MilliporeGS filter (a 2.5 cm disk with a pore size of 0.22 um) prewashed twicewith 0.6 ml of ice-cold TK buffer. The filter was washed with 0.6 ml ofcold TK buffer and dried. The radioactivity retained on the filter wascounted by liquid scintillation, and the results were plotted on a graph(FIG. 13).

d) Preparation of Labelled RBC6

The mixture of 5'-labelling (50 ul) included 500 pmol of the 14 mer, 50mM Tris-HCl (pH7.6), 10 mM MgClhd 2, 5 mM DTT, 50 ug/ul BSA, 20 uM ATP,50 uCi of (gamma-³² P)ATP, 80 units/ml RNasin and 10 units of T4polynucleotide kinase (BioLabs). The reaction was allowed to proceed for1 hr at 37° C. After heating in boiling water for 2 min, the labelled14-mer was purified by 20% polyacrylamide gel electrophoresis, elutedwith water and desalted using a Sephadex NAP-10 column. Annealing to the15-mer was carried out as described above.

e) Binding of RBC6 to Rev Protein

The mixtures for binding (0.5 ml) included 0.6 nM ³² P-labelled RBC6(ca. 20,000 cpm), 24 nM rev protein, varying concentrations (0-150 nM)of unlabelled RBC6 and 80 units/ml RNasin in TK buffer. The filterbinding assays were carried out as described above, and a Scatchard plot(FIG. 14) was obtained from the results. The x intercept shows thestoichiometry of RNA to rev protein, and the K_(d) value is derived fromthe slope.

f) Saturation Binding of Rev Protein to RBC6

The mixtures for binding (0.5 ml) included 1.5 nM ³² P-labelled RBC6(ca. 20,000 cpm), varying concentrations (0-50 nM) of rev protein and 80units/ml RNasin in TK buffer. The filter binding assays were carried outas described above, and the results were plotted on a graph (FIG. 15).

FIG. 16 shows the structures of synthetic oligoribonucleotides which cananneal to form a "bubble" structure stabilized by a "three-way" junctionsimilar to that found in the rev-response element. TWJ-U (SEQ ID NO: 22,SEQ ID NO: 23 and SEQ ID NO: 25) carries an authentic HIV-1_(ARV2)sequence with a G:U base pair formed between G25 and U85. In TWJ-C (SEQID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24) U85 has been replaced by a Cto form a slightly more stable structure. In RBC2-6X only twooligonucleotides are annealed. Although this pair of oligonucleotidescontains all the sequences from the "bubble" region, this structure isnot sufficiently stable to form an effective rev binding site.

FIG. 17 shows competition binding curves for rev binding to thesynthetic ribonucleotides forming the "three-way" junction and "bubble"structures. Results for R7 are indicated by empty squares with dots. TheTWJ-C (SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24) (filled squareswith dots) and TWJ-U (SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 25)(filled diamonds with dots) junctions both compete for rev binding withthe same affinity as the "bubble" structure created by the RBC6 (filleddiamonds) binding site. By contrast, RBC2, 6X (filled squares), whichcontains only two oligonucleotides is unable to compete effectively.Thus in order to act as a rev binding site the sequences comprising"bubble" structure must be stabilized by flanking secondary using eitherdouble-helical structural elements as in RBC6 or a three-way junction asin TWJ-C and TWJ-U. In addition to demonstrating that syntheticthree-way sequences can act as a binding site for rev, these data alsoprovide further support for the model for the secondary structure of therev-responsive element as shown in FIGS. 3 and 4b. In the predictedstructure, the "bubble" sequence is also stabilized by a three-wayjunction.

FIG. 18 shows electron micrographs of filaments formed by rev proteinalone (panel a) or in complexes with the 22 nucleotide long RRE RNAtranscript (panel b) or in complexes with a 2.4 kb transcriptcorresponding to env mRNA. Samples were negatively stained with uranylacetate (120,000 X).

At concentrations above 100 ug/ml rev polymerizes in low salt buffers(50 mM NaCl) and forms a gel (33). Electron micrographs show that thesegels contain large filaments about 14 nm wide and up to 1,500 nm long.Filament formation is temperature dependent, and the longest filamentsare grown by slowly increasing the temperature from 4° C. to 25° C. overa period of several hours, suggesting that filament formation is anentropically driven process that is dependent upon hydrophobicinteractions between rev molecules. The structure of the rev-containingfilaments is fairly regular, with units which are spaced atapproximately 4 nm. There is a band of negative stain running down themiddle of the filaments, suggesting that they are hollow tubes.

When an excess of rev is mixed with the 238-long RRE 7 transcript, shortrod-like ribonucleoprotein complexes with a preferred filament length of60 nm are formed. Filaments as long as 500 to 700 nm have been detectedwhen a 2.4 kb transcript of the env gene is used as a template. Theratios of these two filament lengths suggests the RNA molecules arecoated throughout their entire lengths. The samples examined by electronmicroscopy were also analyzed by sucrose gradient centrifugation and, inthe case of the 238 RRE RNA fragment, by non-denaturing gelelectrophoresis (33). All the RNA transcripts were bound by rev andproduced high molecular weight complexes that could be easilydistinguished from free RNA and were protected from digestion bymicrococcal nuclease (data not shown).

Method

Protein filaments were grown from solutions containing 20 to 100 ug/mlrev protein in 20 mM Tris-HCl pH7.4, 50 mM NaCl, 1.0 mM DTT by slowwarming from 4° C. to 25° C. for a period of several hours. Proteinfilaments are not formed with rev exposed to Triton X-100. Complexesbetween rev protein and RNA transcripts carrying an RRE sequence at the5' end were formed using 0.01 to 0.1 ug/ml RNA, and 22 to 110 ug/ml revprotein in 20 mM Tris-HCl pH7.4, 50 mM NaCl, 0.1 mM EDTA, 1.0 mM DTT, 20units/ml RNasin at 30° C. for 1 hour. Complex formation was monitored bygel mobility shift assays (7), surcrose gradient centrifugation andelectron microscopy. Filaments were negatively stained with uranylacetate and photographed at a magnification of about 50,000 X.

Discussion

The complex binding behaviour of rev has led to some confusion as towhether rev recognises a secondary structure or a specific sequencefeature in the RRE RNA (92,73,97). The work reported here demonstratesthat the RRE contains a purine-rich "bubble" which acts as the highaffinity rev-binding site. However, because of its ability topolymerize, rev is also able to bind RNA sequences adjacent to the highaffinity site. The binding of rev to those lower affinity sites isresponsible for the non-linear Scatchard plots as well as for theformation of progressively larger complexes between rev and RRE RNA asrev concentrations are increased. When rev concentrations aresufficiently high, RNA is packaged into long ribonucleoproteinfilaments, which can easily be detected by electron microscopy. Thus,rev binding to the high affinity site within the RRE RNA may beconsidered to be the nucleation event for an assembly process duringwhich RNA is packaged into filamentous coats. An analogous processoccurs in the packaging of the RNA of tobacco mosaic virus (TMV) andother RNA viruses by their coat proteins (98).

Rev recognition of the "bubble" structure involves both the bulgednucleotides and the two adjacent base pairs on each side. All mutationsknown to abolish rev activity in vivo (51,68,73) are expected to eitherdelete or disrupt the "bubble" sequence. Residues within the "bubble"are highly conserved in different HIV-1 strains, with the exception ofU60 which tolerates C or G substitutions (93). Changes at residue 60 arenot expected to impair RRE function significantly, since we have shownthat the U60 to C substitution produces only a 2-fold reduction in revbinding.

The "bubble" sequence is highly resistant to nuclease cleavage as wellas to modification by chemical reagents (92), suggesting that it forms acompact and rigid structure which locally distorts a double-stranded RNAhelix. Details of the structure are still unknown, but the "bubble"could perhaps be stabilized by a non-Watson-Crick G:A base pair (99)between G34 and A61, as well as by stacking interactions.

RNAs carrying RRE sequences are efficiently packaged in vitro intorod-like filaments which can extend over many hundreds of nucleotidesand coat the entire length of a template RNA molecule. Filamentformation is facilitated by the presence of an RRE sequence. However,rev is also able to bind non-specifically to RNA molecules withapproximately 20 fold lower affinity (33,34). The non-specific bindingof rev allows RNA molecules that do not carry RRE sequences, such as TMVRNA, to be also packaged into filaments in vitro, provided both the revand RNA concentrations are sufficiently high. The intracellular bindingreaction is likely to involve a competition between rev and hnRNPparticle proteins, and this may restrict filament formation to theRRE-containing RNAs.

The RNA binding properties of rev strongly suggest that is blockssplicing simply by packaging unspliced RNA transcripts containing theRRE sequence into inaccessible ribonucleoprotein complexes. Confirmationof our proposal will require the isolation of complexes containing revand viral mRNAs from infected cells. However, there is already indirectevidence in support of an RNA packaging model for rev activity. Rev isable to influence splicing when the RRE is placed either in an intron orin an unspliced exon and when the RRE is placed at various distancesfrom splice sites (46,65). In addition, rev is believed to disruptspicing in vitro by blocking spliceosome formation (46). Finally, the invivo activity of rev is believed to be highly concentration dependentbecause rev-minus viruses can only be rescued by high concentration oftransfected rev-expressing plasmids (88).

The packaging model also provides a simple kinentic explanation for thedelayed appearance of the virion RNA relative to the 4.3 kb mRNAs, suchas the env mRNA (59). Since the RRE sequence is only 535 nucleotidesfrom the splice acceptor sequence for the second exons of the tat andrev genes, only a short rev filament would be needed to block splicingat this site and allow the production of the 4.3 kb mRNAs. Protection ofthe additional unused splice donor and acceptor sites located between1.8-2.0 kb towards the 5' end of the virion RNA would require either theformation of longer ribonucleoprotein filaments or the nucleation offilament formation by rev on secondary sites. In either ease, theseprocesses would be expected to be more efficient towards the end of aninfectious cycle when intracellular rev protein concentrations might beexpected to be maximal.

Although our model implies that the physical properties of rev canaccount for its biological activity, it is possible that cellularco-factor(s) are also required (89). Mouse cells infected by HIV have arev-minus phenotype which can be reversed after fusion to human cells(89). However, in these experiments rev protein levels in the differentcell lines were not measured, and it is possible that less rev wasexpressed in the mouse cells than in the human cells. By contrast, revis functional in Drosphila melanogaster cells (90).

In conclusion we note that the assembly of rev protein on viral mRNAscarrying RRE sequences is a primary event in the HIV life-cycle andthrefore constitutes an important target for therapeutic intervention.As described here, small molecules that interfere with rev binding tothe "bubble" sequence can be expected to show anti-HIV activity.

                  TABLE I                                                         ______________________________________                                        Mutagenesis of the rev binding site.                                                                     K.sub.d                                                                              % RNA bound                                   Mutation (nM) (10 nM rev)                                                   ______________________________________                                        A. Normal rev binding                                                           R7      wild-type            3    50                                          R33 wild-type 3 40                                                            R57 G38: C57 → A: U 3 40                                             B. Reduced rev binding                                                          R35     ΔG35           4    35                                          R50 G36 → A 5 20                                                       R52 U60 → C 5 18                                                       R39 U33: A63 → A: U 8 15                                               R54 U33: A63 → C: G 6 17                                               R55 G34: C62 → A: U 4 21                                               R42 G38: C57 → C: G 8 18                                             C. Non-specific rev binding                                                     R49     ΔG59           --   2                                           R45 ΔU60 -- 6                                                           R46 ΔA61 -- 1                                                           R34 ΔG35-G36 -- 3                                                       R36 ΔG59-A61 -- 4                                                       R58 G59 → A -- 6                                                       R53 A61 → G -- 5                                                       R37 G34, G35, G36 → A, A, A; C62 → U -- 5                       R47 G35 → A -- 6                                                       R40 G34: C62 → C: G -- 3                                               R56 C37: G58 → U: A -- 6                                               R41 C37: G58 → G: C -- 2                                             ______________________________________                                    

Filter binding assays contained 20 pg uniformly labelled RNA probe (500dpm per pg RNA), 1 μg salmon sperm DNA, 0.45 μg yeast tRNA and 40 unitsRNasin (Promega) in 500 μl buffer containing 43 mM Tris-HCl pH 8.0, 50mM KCl. Mutations in the purine-rich "bubble" sequence (numbered asshown in FIG. 2) were introduced into the R33 stem-loop structure (FIG.3d) by site-directed mutagenesis.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 26                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 209                                                             <212> TYPE: RNA                                                               <213> ORGANISM: Human immunodeficiency virus                                   - - <400> SEQUENCE: 1                                                         - - ccuuggguuc uugggagcag caggaagcac uaugggcgca gccucaauga cg -            #cugacggu     60                                                                 - - acaggccaga caauuauugu cugguauagu gcagcagcag aacaauuugc ug -            #agggcuau    120                                                                 - - ugaggcgcaa cagcaucugu ugcaacucac agucuggggc aucaagcagc uc -            #caagcaag    180                                                                 - - aguccuagcu guggaaagau accuaaagg         - #                  - #               209                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 66                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 2                                                         - - gcacuauggg cgcagcguca augacgcuga cgguacaggc cagacaauua uu -             #gucuggua     60                                                                 - - uagugc                 - #                  - #                  -     #           66                                                                  - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 111                                                             <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 3                                                         - - gggagaccgg aauucccuug gguucuuggg agcagcagga agcacuaugg gc -             #gcaguguc     60                                                                 - - auugacgcug acgguacagg ccagacaauu auugucuggu auagugcaac a - #                111                                                                        - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 83                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 4                                                         - - gggagaccgg aauucagcac uaugggcgca gugucauuga cgcugacggu ac -             #aggccaga     60                                                                 - - caauuauugu cugguauagu gca           - #                  - #                    83                                                                     - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 67                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 5                                                         - - gggagaccgg aauucagcac uagggcgcag ugucauugac gcugagggua ca -             #ggccagac     60                                                                 - - aauuaua                 - #                  - #                       - #          67                                                                  - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 50                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic           RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 6                                                         - - gggagaccgg aauucugggc gcagugucau ugacgcugac gguacaggca  - #                  50                                                                         - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 49                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 7                                                         - - gggagaccgg aauucugggc gcagugucau ugacgcugcg guacaggca  - #                   49                                                                         - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 50                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 8                                                         - - gggagaccgg aauucugggc gcagugucaa ugacgcugac gguacaggca  - #                  50                                                                         - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 49                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 9                                                         - - gggagaccgg aauucugggc gcagcgucau ugacacugcg guacaggca  - #                   49                                                                         - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 36                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 10                                                        - - gggagaccgg aauucugggc gcuucggcgg uacaga      - #                  -     #       36                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 56                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 11                                                        - - gggagaccgg aauucugggc gcagugucau ugacgcugcg guacagaauu cc - #ggca             56                                                                        - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 56                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 12                                                        - - gggagaccgg aauucuaaac gcagugucau ugacgcugcg guauagaauu cc - #ggca             56                                                                        - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 55                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 13                                                        - - gggagaccgg aauucuggcg cagugucauu gacgcugcgg uacagaauuc cg - #gca              55                                                                        - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 53                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 14                                                        - - gggagaccgg aauucugggc gcagugucau ugacgcugcg cagaauuccg gc - #a                53                                                                        - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 53                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 15                                                        - - gggagaccgg aauucugcgc agugucauug acgcugcggu acagaauucc gg - #c                53                                                                        - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 10                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 16                                                        - - ugugggcgca                - #                  - #                      - #        10                                                                   - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 10                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 17                                                        - - ugcgguacac                - #                  - #                      - #        10                                                                   - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 12                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 18                                                        - - gugugggcgc ag              - #                  - #                      - #       12                                                                   - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 12                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 19                                                        - - cugcgguaca ca              - #                  - #                      - #       12                                                                   - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 14                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 20                                                        - - cgugugggcg cagc              - #                  - #                      - #     14                                                                   - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 15                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 21                                                        - - gcugcgguac acacg              - #                  - #                      - #    15                                                                   - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 16                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 22                                                        - - gcacuauggg cgcagc             - #                  - #                      - #    16                                                                   - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 18                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 23                                                        - - gcugcgguac aggccaga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 13                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 24                                                        - - ucuggcauag ugc              - #                  - #                      - #      13                                                                   - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 13                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 25                                                        - - ucugguauag ugc              - #                  - #                      - #      13                                                                   - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 15                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic            RNA derived from HIV RRE sequence                                        - - <400> SEQUENCE: 26                                                        - - gggagaccgg aauuc              - #                  - #                      - #    15                                                                 __________________________________________________________________________

What is claimed is:
 1. An assay for identifying a compound that inhibitsthe binding of HIV Rev to HIV RRE, comprisingcontacting a candidatecompound with Rev in the presence of an oligonucleotide that binds toHIV Rev protein with an affinity of no more than 8 nM, wherein thesequence of said oligonucleotide is not found in wild type RRE, andwherein said oligonucleotide comprises a purine-rich bubble containingWatson-Crick base-paired and non-base-paired nucleotides; anddetermining the degree of binding of Rev to said oligonucleotiderelative to the binding in the absence of said candidate compound.
 2. Anassay for identifying a compound that inhibits the binding of HIV Rev toHIV RRE, comprisingcontacting a candidate compound with Rev in thepresence of an oligonucleotide that binds to HIV Rev protein, saidoligonucleotide comprising a purine-rich bubble containing Watson-Crickbase-paired and non-base-paired nucleotides and said oligonucleotidecomprising at least one base pair adjacent to said bubble and not foundin wild type RRE, on at least one side of said bubble; and determiningthe degree of binding of Rev to said oligonucleotide relative to thebinding in the absence of said candidate compound.
 3. The assay of claim2 wherein said oligonucleotide comprises at least two adjacent basepairs on at least one side of said bubble.
 4. The assay of claim 2wherein said oligonucleotide comprises at least four adjacent base pairson at least one side of said bubble.
 5. The assay of claim 1 or 2,wherein the sequence of said purine-rich bubble is selected from thegroup consisting of: ##STR1##
 6. The assay of claim 1 or 2, wherein saidoligonucleotide comprises one or more 2'-deoxyribonucleotides outside ofsaid purine-rich bubble.
 7. An assay for identifying a compound thatinhibits the binding of HIV Rev to HIV RRE, comprising contacting acandidate compound with Rev in the presence of a first oligonucleotideand a second oligonucleotide, wherein said first and secondoligonucleotides form a duplex that binds to HIV Rev protein with anaffinity of no more than 8 nM, and said duplex comprises a purine-richbubble containing Watson-Crick base-paired and non-base-pairednucleotides; anddetermining the degree of binding of Rev to saidannealed first oligonucleotide and second oligonucleotide relative tothe binding in the absence of said candidate compound.
 8. An assay foridentifying a compound that inhibits the binding of HIV Rev to HIV RRE,comprisingcontacting a candidate compound with Rev in the presence of afirst oligonucleotide and a second oligonucleotide, wherein said firstand second oligonucleotides form a duplex that binds to HIV Rev proteinand comprises a purine-rich bubble containing Watson-Crick base-pairedand non-base-paired nucleotides and said duplex comprises at least onebase pair adjacent to said bubble and not found in wild type RRE, on atleast one side of said bubble; and determining the degree of binding ofRev to said annealed first oligonucleotide and second oligonucleotiderelative to the binding in the absence of said candidate compound. 9.The assay of claim 8, wherein said oligonucleotide comprises at leasttwo adjacent base pairs on at least one side of said bubble.
 10. Theassay of claim 8, wherein said oligonucleotide comprises at least fouradjacent base pairs on at least one side of said bubble.
 11. The assayof claim 7 or 8, wherein said first oligonucleotide comprises thesequence 5'-UGGGCG-3' and is annealed to said second oligonucleotidecomprising the sequence 5'-CGGUACA-3' to form said purine-rich bubble,which corresponds to ##STR2## or said first oligonucleotide comprisesthe sequence 5'-UGGGCA-3' and is annealed to said second oligonucleotidecomprising the sequence 5'-UGGUACA-3' to form said purine-rich bubble,which corresponds to ##STR3## or said first oligonucleotide comprisesthe sequence 5'-UGGGCU-3' and is annealed to said second oligonucleotidecomprising the sequence 5'-AGGUACA-3' to form said purine-rich bubble,which corresponds to ##STR4## or said first oligonucleotide comprisesthe sequence 5'-UGGGCG-3' and is annealed to said second oligonucleotidecomprising the sequence 5'-CGGCACA-3' to form said purine-rich bubble,which corresponds to ##STR5## or said first oligonucleotide comprisesthe sequence 5'-UGGGCG-3' and is annealed to said second oligonucleotidecomprising the sequence 5'-CGGGACA-3' to form said purine-rich bubble,which corresponds to ##STR6##
 12. The assay of claim 7 or 8, whereineither said first oligonucleotide or said second oligonucleotidecomprises one or more 2'-deoxyribonucleotides outside of saidpurine-rich bubble.